Use of Combination of Anti-PD-1 Antibody and VEGFR Inhibitor in Preparation of Drug for Treating Cancers

ABSTRACT

Disclosed is the use of a combination of an anti-PD-1 antibody and a VEGFR inhibitor in the preparation of a drug for treating cancers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/339,819, filed Apr. 5, 2019 (now allowed), which claims the benefitof International Application No. PCT/CN2017/105410, filed Oct. 9, 2017,which was published in the Chinese language on Apr. 19, 2018, underInternational Publication No. WO 2018/068691 A1, which claims priorityunder 35 U.S.C. § 119(b) to Chinese Application No. 201610884688.3,filed Oct. 10, 2016. The disclosure of each of these prior applicationsis incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing that is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “065825-91US2 Sequence Listing” and a creation date of Oct.21, 2021, and having a size of 7.28 kb. The sequence listing submittedvia EFS-Web is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the use of a combination of ananti-PD-1 antibody and a VEGFR inhibitor in the preparation of amedicament for the treatment of cancer.

BACKGROUND OF THE INVENTION

PD-1 antibodies specifically recognize and bind to PD-1 on the surfaceof lymphocytes, which leads to the blockade of PD-1/PD-L1 signalingpathway. This in turn activates the immune cytotoxicity of T cellsagainst tumors and modulates the immune system of the body to eliminatetumor cells in vivo. WO201508584 discloses a novel anti-PD-1 antibody,which is currently in clinical trials and has shown an anti-tumoreffect.

Apatinib is the first oral anti-angiogenic drug for advanced gastriccancer in the world, which is highly selective for VEGFR-2 and has apotent anti-angiogenic effect. In a multicenter, randomized,double-blind, placebo-controlled phase III clinical trial of apatinib inpatients with metastatic gastric/gastroesophageal junction cancer afterreceiving second line therapy, the results showed that, when comparedwith placebo, apatinib alone could prolong median overall survival by1.8 months, median progression-free survival by 0.8 months, and adverseevents were controllable (Randomized, Double-Blind, Placebo-ControlledPhase III Trial of apatinib in Patients With Chemotherapy-RefractoryAdvanced or Metastatic Adenocarcinoma of the Stomach or GastroesophagealJunction. J Clin Oncol, 2016 Feb. 16). The structural formula ofapatinib is as shown in formula (I).

CN101676267A discloses a series of salts of apatinib, such as mesylate,hydrochloride, and the like. The pre-clinical animal experimentsdisclosed in CN101675930A also show that apartinib combined withcytotoxic drugs such as oxaliplatin, 5-Fu, docetaxel and doxorubicin cansignificantly improve the therapeutic effect.

At present, no combinational use of a PD-1 antibody and a VEGFRinhibitor has been approved for marketing, but multiple PD-1 antibodies(from other companies) and VEGFR inhibitors (such as sunitinib,sorafenib, etc.) are in phase II/III clinical trial, and the indicationsare respectively malignant liver cancer (sorafenib combined with PD-1antibody) and metastatic renal cell carcinoma (sunitinib combined withPD-1 antibody). The preliminary results showed that the combinations oftwo drugs are more effective and better than the single drug.

WO2015119930 discloses the use of a PD-1 antibody in combination withaxitinib, and WO2015088847 discloses the use of a PD-1 antibody incombination with pazopanib. However, the action mechanism of these VEGFRinhibitors, including sorafenib, sunitinib, axitinib and pazopanib,differ from that of apatinib. Apatinib has the strongest inhibitoryeffect on VEGFR-2, but it has little or no inhibition on other kinases,that is, apatinib is highly selective for VEGFR-2. Therefore, thedisease treated by apatinib is also different from the aforementioneddrugs, and whether apatinib can synergize with a PD-1 antibody andimprove its efficacy need to be further studied. In addition, accordingto a current clinical study of a PD-1 antibody administered alone (PhaseI study of the anti-PD-1 antibody SHR-1210 in patients with advancedsolid tumors. (2017): e15572-e15572), the incidence of capillaryhemangioma was as high as 79.3%, the incidence of hypothyroidism was29.3%, the incidence of pruritus was 19.0%, and the incidence ofdiarrhea was 10.3%. Such high incidence of adverse effects undoubtedlyput a burden on the mental health and quality of life of cancerpatients; therefore, it is very important to reduce adverse effectsduring drug administration.

SUMMARY OF THE INVENTION

The present invention provides use of a combination of an anti-PD-1antibody and a VEGFR inhibitor in the preparation of a medicament forthe treatment of cancer.

Preferably, the VEGFR inhibitor is a VEGFR-2 inhibitor.

A preferred VEGFR inhibitor of the present invention is a VEGFRinhibitor which has an IC50 of less than 100 nM for VEGFR kinase and hasno inhibitory activity against EGFR, HER2, FGFR (IC50>10000 nM),according to the test method disclosed in CN101676267A. A particularlypreferred VEGFR inhibitor is a VEGFR-2 inhibitor having an IC50 of lessthan 50 nM for VEGFR-2 kinase, preferably less than 20 nM, morepreferably less than 10 nM, and most preferably less than 5 nM, and theinhibitory effect thereof on VEGFR-1 or VEGFR-3 is poor, for example,its IC50 is greater than 20 nM, preferably greater than 50 nM.

In a preferred embodiment of the present invention, the VEGFR-2inhibitor is apatinib or a pharmaceutically acceptable salt thereof.

The anti-PD-1 antibody is known, and preferably the light chain variableregion of the anti-PD-1 antibody comprises LCDR1, LCDR2 and LCDR3 asshown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

The heavy chain variable region of the anti-PD-1 antibody comprisesHCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ IDNO: 3, respectively.

Wherein the CDR sequences described above are shown in the followingtable:

Name Sequence NO. HCDR1 SYAWS SEQID NO: 1 HCDR2 TISGGGANTYYPDSVKGSEQID NO: 2 HCDR3 QLYYFDY SEQID NO: 3 LCDR1 LASQTIGTWLT SEQID NO: 4LCDR2 TATSLAD SEQID NO: 5 LCDR3 QQVYSIPWT SEQID NO: 6

Preferably, the anti-PD-1 antibody is a humanized antibody.

The preferred humanized antibody light chain sequence is the sequence asshown in SEQ ID NO: 8 or a variant thereof; the variant preferably has0-10 amino acid substitution(s) in the light chain variable region; morepreferably, has the amino acid change of A43 S.

The humanized antibody heavy chain sequence is the sequence as shown inSEQ ID NO: 7 or a variant thereof; the variant preferably has 0-10 aminoacid substitution(s) in the heavy chain variable region; morepreferably, has the amino acid change of G44R.

Particularly preferred, the humanized antibody light chain sequence isthe sequence as shown in SEQ ID NO: 8, and the heavy chain sequence isthe sequence as shown in SEQ ID NO: 7.

The sequences of the aforementioned humanized antibody heavy and lightchains are as follows:

Heavy chain SEQ ID NO: 7EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYMMSWVRQAPGKGLEWVATISGGGANTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQLYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK Light chainSEQ ID NO: 8 DIQMTQSPSSLSASVGDRVTITCLASQTIGTWLTWYQQKPGKAPKLLIYTATSLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVYSIPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

In a preferred embodiment of the present invention, the VEGFR inhibitorcan also be selected from the group consisting of MP-0250, DE-120,ALN-VSP, Aflibercept, Anecortave, BI-695502, Bevacizumab, PF-06439535,Carboxyamidotriazole, Vanucizumab, RG-7716, Bevacizumab analogue,Navicixizumab, Ranibizumab, Ranibizumab analogue, Conbercept, IBI-302,BI-836880, ARQ-736, RPI-4610, LMG-324, PTC-299, ABT-165, AG-13958,Brolucizumab, PAN-90806, Vatalanib, ODM-203, Altiratinib, TG-100572,OPT-302, TG-100801, CEP-7055, TAS-115, Ilorasertib, Foretinib,JNJ-26483327, Metatinib, R-1530, Tafetinib, Vorolanib, Donafenib,Subutinib, Regorafenib, VGX-100, ENMD-2076, Anlotinib, Ningetinib,Tesevatinib, Tanibirumab, Lucitanib, Cediranib, Chiauranib, IMC-3C5,Glesatinib, KRN-633, Icrucumab, PF-337210, RAF265, Puquitinib,SU-014813, Tivozanib, Fruquintinib, Sitravatinib, Pegaptanib, Pazopanib,Vandetanib, Axitinib, Sulfatinib, Ramucirumab, Plitidepsin, Orantinib,Alacizumab pegol, Telatinib, Ponatinib, Cabozantinib, Lenvatinib,Brivanib Alaninate, and Linifanib.

In the use of the present invention, the cancer is preferably a cancerexpressing PD-L1; more preferably is breast cancer, lung cancer, gastriccancer, intestinal cancer, renal cancer, liver cancer, melanoma, ornon-small cell lung cancer; most preferably is non-small cell lungcancer, melanoma and kidney cancer, or intestinal cancer, and theintestinal cancer includes colon cancer, colorectal cancer, and thelike.

Apatinib is preferably administered in the form of pharmaceuticallyacceptable salt when being administered. The pharmaceutically acceptablesalt can be selected from the group consisting of mesylate andhydrochloride.

Specifically, when being administered, the anti-PD-1 antibody can beadministered at a dose of 0.5-30 mg/kg, preferably 2-10 mg/kg, morepreferably 2-6 mg/kg, and most preferably 3 mg/kg It can be administeredonce every 1 to 3 weeks, preferably once every 2 weeks. For adulthumans, a fixed dose can also be used, for example 100-1000 mg peradministration, preferably 200-600 mg. The dose of the VEGFR inhibitorcan be 3-200 mg/kg. For adult humans, a fixed dose can also be used, forexample 100-1000 mg, 250-1000 mg, preferably 400-850 mg, 100-500 mg, itcan be administered once per day.

In the present invention, the term “combination” is a mode ofadministration, including various situations, in which two drugs areadministered sequentially or simultaneously. Herein, so-called“simultaneously” refers to the administration of the anti-PD-1 antibodyand the VEGFR inhibitor during the same administration cycle. Forexample, administration of the two drugs within 2 days or within 1 day.So-called “sequentially” administrated includes the administration ofthe anti-PD-1 antibody and the VEGFR inhibitor in differentadministration cycles, respectively. These modes of administration allbelong to the combination administration described in the presentinvention.

In a preferred embodiment of the present invention, the anti-PD-1antibody is administered by injection, for example, subcutaneously orintravenously, and the anti-PD-1 antibody is formulated in an injectableform prior to injection. A particularly preferred injectable form of theanti-PD-1 antibody is injection or a lyophilized powder comprising theanti-PD-1 antibody, buffer, stabilizer, and optionally comprisingsurfactant. The buffer can be selected from one or more of acetate,citrate, succinate and phosphate. The stabilizer can be selected fromsugars or amino acids, preferably disaccharide such as sucrose, lactose,trehalose and maltose. The surfactant is selected from the groupconsisting of polyoxyethylene hydrogenated castor oil, glycerin fattyacid ester, polyoxyethylene and sorbitan fatty acid ester; preferablythe polyoxyethylene sorbitan fatty acid ester is polysorbate 20, 40, 60or 80; most preferred is polysorbate 20. The most preferred injectableform of the anti-PD-1 antibody comprises anti-PD-1 antibody, acetatebuffer, trehalose and polysorbate 20.

The present invention provides the anti-PD-1 antibody as described abovein combination with the VEGFR as described above, as a medicament fortreating tumors.

The present invention provides the anti-PD-1 antibody as described abovein combination with the VEGFR as described above as a medicament forreducing adverse effect of drugs. Preferably, the adverse effect ofdrugs is selected from the effect caused by the anti-PD-1 antibody orthe VEGFR inhibitor.

The present invention provides the anti-PD-1 antibody as described abovein combination with the VEGFR inhibitor as described above, as amedicament for reducing the dose of the anti-PD-1 antibody administeredalone and/or the dose of the VEGFR inhibitor administered alone.

The present invention provides a method for treating tumors/cancercomprising administering to a patient the anti-PD-1 antibody asdescribed above and the VEGFR inhibitor as described above.

The present invention provides a method for reducing the dose of eitherthe anti-PD-1 antibody or the VEGFR inhibitor administered alone,comprising administering to a patient the anti-PD-1 antibody asdescribed above in combination with the VEGFR inhibitor as describedabove.

Preferably, when administered in combination with the anti-PD-1antibody, the VEGFR inhibitor is administered at a dose of 10% to 100%,preferably 10% to 75%, more preferably 75%, 50%, 25%, 12.5% of the doseadministered alone.

Preferably, when administered in combination with the VEGFR inhibitor,the anti-PD-1 antibody is administered at a dose of 10% to 100%,preferably 10% to 50% of the dose administered alone.

In a preferred embodiment of the present invention, when the anti-PD-1antibody is administered in combination with the VEGFR inhibitor, theadverse effect of drugs mediated by the anti-PD-1 antibody and/or theimmune system can be reduced; preferably, the adverse effect is selectedfrom the group consisting of a vascular-associated adverse effect,glandular hypofunction, skin adverse effect, respiratory system adverseeffect, liver-associated adverse effect, endocrine-associated adverseeffect, digestive system adverse effect, kidney-associated adverseeffect, fatigue, and pyrexia. The preferred vascular-associated adverseeffect is selected from the group consisting of hemangioma, vasculitis,and lymphangioma; the glandular hypofunction is selected from the groupconsisting of hypothyroidism, hypoparathyroidism, pancreatichypofunction, and prostatic hypofunction; the skin adverse effect isselected from the group consisting of pruritus, urticaria, rash, andtoxic epidermal necrosis; the respiratory adverse effect is selectedfrom the group consisting of pneumonia, bronchitis, chronic obstructivepulmonary disease, and pulmonary fibrosis; the liver-associated adverseeffect is selected from the group consisting of hepatitis and liverdysfunction; the endocrine-associated adverse effect is selected fromthe group consisting of diabetes type I, diabetes type II, andhypoglycemia; the kidney-associated adverse effect is selected from thegroup consisting of nephritis and renal failure; the digestive systemadverse effect is selected from the group consisting of diarrhea,nausea, emesis, enteritis, and constipation. More preferably, theadverse effect of drugs is selected from the group consisting ofhemangioma, hypothyroidism, hypoparathyroidism, pruritus, pneumonia,hepatitis, liver dysfunction, diabetes type I, nephritis, and renalfailure.

The present invention provides a pharmaceutical kit or a pharmaceuticalpackage, comprising the VEGFR inhibitor and the anti-PD-1 antibody asdescribed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of administration of antibody and compound on therelative volume of MC38 (PD-L1) xenograft in tumor-bearing mice.

FIG. 2. Effect of administration of antibody and compound on body weightof tumor-bearing mice with MC38 (PD-L1) xenograft, wherein * indicatesp<0.05, vs blank control group.

FIG. 3. Effect of administration of antibody and compound on MC38(PD-L1) xenograft in tumor-bearing mice—tumor weight.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described below in conjunction with theexamples. These examples are not intended to limit the scope of thepresent invention.

Example 1: Effect of the Anti-PD-1 Antibody and Apatinib Mesylate,Administered Alone or in Combination, on Human PD-1 Transgenic C57 Micewhich Bear Mouse Colon Cancer Cell MC-38 (PD-L1) Xenograft Transferredwith PD-L1 Gene

1. Study purposes: Human PD-1 transgenic mice were used as test animals.The effects of anti-PD-1 antibody in combination with apartinib on humanPD-1 expressed in transgenic C57 mice were evaluated, and the transgenicmice bear mouse colon cancer cell MC-38 (PD-L1) xenograft transfectedwith PD-L1 gene.

2. Test antibodies and compounds: The anti-PD-1 antibody was preparedaccording to the method disclosed in WO2015085847 in which thecorresponding code of the antibody is H005-1, and the sequences of theheavy and light chain are shown in SEQ ID NO: 7 and SEQ ID NO: 8 in thepresent invention. Lot number: P1512, 200 mg/vial, formulated into 20mg/ml before use. Apatinib mesylate was prepared according to the methoddisclosed in CN101676267A, lot number: 668160401; molecular weight:493.58; purity: 99.60%.

3. Experimental animals: Human PD-1 transgenic C57 mice,specific-pathogen-free (SPF), with different body weights, 50% male and50% female, were purchased from IsisInnovation Limited, UK.

4. Drug preparation: Anti-PD-1 antibody (3 mg/kg): anti-PD-1 antibodystock solution (20 mg/ml) was adjusted to a concentration of 0.3 mg/mlwith phosphate buffered saline (PBS), and the intraperitoneal injectionvolume was 0.2 ml/mouse.

Apatinib (200 mg/kg): 400 mg apatinib was dissolved in 20 ml of 0.5%sodium carboxymethylcellulose (NaCMC), adjusted to 20 mg/ml, and wasadministered in 0.2 ml per mouse by gavage.The solvent vehicle (HIgG) control contained human IgG (3 mg/kgdissolved in 0.5% CMC, adjusted to 0.3 mg/ml, and the volume forintraperitoneal injection was 0.2 ml/mouse.

5. Test method:

-   -   5.1 C57 mice were adapted to the laboratory environment for >5        days.    -   5.2 Tumor cells transplantation: Skin preparation was performed        on human PD-1 transgenic C57 mice one day before MC38 (PD-L1)        cells (5×10⁶/mouse) were inoculated subcutaneously at the right        flank on June 12. The tumors were then grown for 8 days. When        the tumors reached 142.17±13.30 mm³, the animals were randomly        assigned to 4 groups (d0) with 8 mice in each group (four male        mice and four female mice in each group).    -   5.3 Dose and method of administration: Anti-PD-1 antibody was        injected intraperitoneally, Q2D*7 (once every 2 days, 7 times in        total), and apatinib was administered by oral gavage, QD*14        (once a day for 14 days). The specific drug administration        regimen is shown in Table 1.    -   5.4 Determination of volume of xenograft and body weight of        mice: Tumor volume and body weight were measured twice a week        and data were recorded.    -   5.5 Statistics: Excel 2003 statistical software was used: the        mean was calculated by avg; the SD value was calculated by        STDEV; the SEM value was calculated by STDEV/SQRT; the P value        indicating the difference between groups is calculated by TTEST.        The formula for calculating tumor volume (V) is:

V=½×L _(long) ×L _(short) ²

Relative volume (RTV)=V _(T) /V ₀

Tumor inhibition rate (%)=(C _(RTV) −T _(RTV))/C _(RTV)(%)

Wherein, V₀ and V_(T) are the tumor volume at the beginning of theexperiment and at the end of the experiment, respectively. C_(RTV) andT_(RTV) are the relative tumor volumes of the blank control group andthe experimental group at the end of the experiment, respectively.

6. Test results: The PD-1 antibody was injected intraperitoneally,Q2D*7, and compound apatinib was administered by oral gavage, QD*14. Onday 21 after the initial administration, the tumor inhibition rate ofthe group administered with the anti-PD-1 antibody (3 mg/kg) alone was20.40%, and the tumor inhibition rate of the group administered withapatinib (200 mg/kg) alone was 35.67%. The tumor inhibition rate of thecombination of the anti-PD-1 antibody (3 mg/kg) and apatinib (200 mg/kg)was 63.07% (significantly different from that in human IgG controlgroup) There were no significant differences between the otheradministration groups (administration of agent alone) relative to thehuman IgG control group. From the experimental results, the efficacy ofthe combination group of the anti-PD-1 antibody (3 mg/kg)+apatinib (200mg/kg) is superior to that of the anti-PD-1 antibody administrated aloneand that of apintinib administrated alone. The body weight of mice ineach group was normal, indicating that the drug had no obvious sideeffects. The specific data are shown in Table 1 and FIGS. 1-3.

Example 2: Clinical Study of Anti-PD-1 Antibody Combined with ApatinibMesylate in the Treatment of Advanced Malignant Tumor

Inclusion criteria: (1) advanced malignancy; (2) failure in chemotherapyby using first-line, second-line or above; (3) measurable lesions; (4)ECOG score 0-1.

Test drugs: commercially available apatinib mesylate tablet; theanti-PD-1 antibody of Example 1.

Method of administration: Up to Sep. 20, 2017, a total of 31 subjectswere screened, 30 subjects were enrolled (14 subjects withdrew fromtreatment, and 16 subjects were still in the group of administration).

Administration method for subjects No. 001-005 was intravenous infusionof anti-PD-1 antibody, 3 mg/kg, once every 2 weeks; apatinib orally, 500mg, once a day. Administration method for subjects No. 006-010 wasintravenous infusion of anti-PD-1 antibody, 200 mg, once every 2 weeks;apatinib orally, 125 mg, once a day. Administration method for subjectsNo. 011-031 was intravenous infusion of anti-PD-1 antibody, 200 mg, onceevery 2 weeks; apatinib orally, 250 mg, once a day.

Clinical Outcome: In terms of effectiveness, in the 6^(th) week, therewere 24 evaluable data for efficacy evaluation, with a disease controlrate (DCR) of 83.3% (20/24); in the 12th week, there were 19 evaluabledata for efficacy evaluation, with a DCR of 63.2% (12/19); in the 18thweek, there were 10 evaluable data for efficacy evaluation, with a DCRof 70% (7/10); in the 24th week, there were 5 evaluable data forefficacy evaluation, with a DCR of 80% (4/5); up to Sep. 20, 2017, therewere 2 hepatocellular carcinoma subjects with their 24-week effect ofpartial response (PR) and progression-free survival (PFS) of more than 6months. Among the 24 evaluable data, there were 4 cases showing optimalefficacy with PR, 15 cases of stable disease (SD), and 5 cases ofprogressive disease (PD). Although the objective response rate (ORR) wasonly 16.7%, the DCR was high, e.g., as high as 79%, and some subjectshad a PFS of more than 6 months. The specific results are shown in Table2, Table 3 and Table 4. In addition, the dose of apatinib alone intreatment of solid tumor (such as gastric cancer, gastroesophagealjunction adenocarcinoma, liver cancer, etc.) was usually up to 850mg/day (see instructions for apatinib). However, in embodiments of theinvention the combination of apatinib and anti-PD-1 antibody makes itpossible to reduce the dose of apatinib down to 125 mg/day, and providesimproved effectiveness and better safety when compared with apatinibadministrated alone. In terms of safety, up to September 20, 11 cases ofserious adverse events (SAE) were reported in 8 subjects, and theincidence of SAE was 26.7% (8/30). Seven of the SAEs were observed insubjects No. 001-005 (wherein the dose of apatinib for initial test washigh, 500 mg) and accounted for most of the serious adverse events.However, with modified dosage regimen, it was found that good anti-tumoreffect could be maintained, and the adverse effects caused by high doseof apintinib could be significantly reduced. In addition, in thisclinical study it was surprisingly found that the combination ofapatinib and anti-PD-1 antibody showed almost no hemangioma-associatedadverse effect in the treatment of malignant tumors when compared withanti-PD-1 antibody alone. Hemangiomas was observed in only one subjectwho was administrated with PD-1 antibody alone, due to intolerance tocombination therapy.

Example 3: Phase II Clinical Study of Anti-PD-1 Antibody Combined withApatinib Mesylate in the Treatment of Advanced Non-Small Cell LungCancer

Inclusion criteria: (1) advanced non-small cell lung cancer; (2) failurein chemotherapy by using first-line or second-line or above; (3)measurable lesions; (4) ECOG score 0-1.

Test drugs: commercially available apatinib mesylate tablet; theanti-PD-1 antibody of Example 1.

Method of administration: Anti-PD-1 antibody, once every 2 weeks,intravenous infusion, 200 mg each time; apatinib mesylate orally, oncedaily, 250 mg or 375 mg or 500 mg each time.

Clinical results: up to July 28, a total of 15 subjects were screened,of which 12 were enrolled. A total of 12 subjects completed at least 1cycle of administration observation, 10 patients (10/12) had disease instable condition, and 1 patient had partial remission. See Table 5 fordetails. Surprisingly, it was found that the combination of apatinibmesylate and anti-PD-1 antibody enhanced the efficacy and reduced theadverse effects when compared with anti-PD-1 antibody administeredalone. In this study, the common adverse effects were usually grade I toII, and the incidence of anti-PD-1 antibody-associated orimmune-associated adverse effects (such as capillary hemangioma) wasonly 8% (1 case), the incidence of hypothyroidism was only 8% (1 case),and gastrointestinal adverse effects (such as diarrhea) and skin adverseeffect (such as pruritus) were not observed. In an ASCO report publishedin 2017, anti-PD-1 antibody administrated alone for the treatment ofsolid tumors in phase I clinical trial exhibited an incidence ofcapillary hemangioma as high as 79.3%, and the incidence ofhypothyroidism was 29.3%, the incidence of pruritus was 19.0%, theincidence of diarrhea was 10.3% (Phase I study of the anti-PD-1 antibodySHR-1210 in patients with advanced solid tumors. (2017): e15572-e15572).Therefore, the combination of apatinib mesylate and anti-PD-1 antibodycan not only alleviate or control the tumor proliferation of non-smallcell lung cancer (which has experienced chemotherapy failure), but alsoreduce the anti-PD-1 antibody-associated or immune-mediated adverseeffects and improve the life quality of patients.

TABLE 1 Number Mean tumor volume Mean tumor volume Relative tumor %Tumor of (mm³) (mm³) volume inhibition P animals/ Group AdministrationRoute D 0 SEM D 21 SEM D 21 SEM rate D 21 (vs blank) group HIgG (3mg/kg) Q2D*7 ip 141.46 13.23 1983.55 292.09 14.41 2.07 — — 8 Anti-PD-1antibody Q2D*7 ip 146.40 12.68 1652.93 309.61 11.47 2.49 20.40% 0.3791648 (3 mg/kg) Anti-PD-1 antibody (3 Q2D*7/ ip/po 146.11 11.69 771.95 73.425.32 0.73 63.07%** 0.001007 8 mg/kg) + apatinib QD(14 D) (200 mg/kg)apatinib QD(14 D) po 139.70 7.59 1263.86 206.54 9.27 1.58 25.67%0.068923 8 **p < 0.01, vs control group

TABLE 2 Administration methods: PD-1 antibody 3 mg/kg + apatinib 500 mgPrevious Treatment 6 weeks 12 weeks 18 weeks 24 weeks 32 weeks OptimalNo. Diagnosis therapy cycle evaluation evaluation evaluation evaluationevaluation efficacy 001 gastric cancer Second-line 1 NA NA NA NA NA Notevaluated therapy 002 gastric cancer Fouth-line 6 SD reduced PD NA NA NASD therapy 003 gastric cancer Fifth-line 9 SD reduced SD reduced PD PDNA SD therapy 004 hepatocellular First-line 2 SD increased NA NA NA NASD carcinoma therapy 005 hepatocellular Second-line 1 NA NA NA NA NA Notevaluated carcinoma therapy

TABLE 3 Administration methods: PD-1 antibody 200 mg + apatinib 125 mgPrevious Treatment 6 weeks 12 weeks 18 weeks 24 weeks 32 weeks OptimalNo. Diagnosis therapy cycle evaluation evaluation evaluation evaluationevaluation efficacy 006 hepatocellular Second-line 18 SD increased SDincreased PD PR SD PR carcinoma therapy 007 hepatocellular Second-line18 SD SD reduced SD reduced SD Performed, SD carcinoma therapy Notevaluated 009 hepatocellular Second-line 18 SD SD reduced SD reduced SDreduced NA SD carcinoma therapy 008 hepatocellular First-line 4 PD NA NANA NA PD carcinoma therapy 010 gastric cancer Third-line 2 NA NA NA NANA Not evaluated therapy

TABLE 4 Administration methods: PD-1 antibody 200 mg + apatinib 250 mgTreatment 6 weeks 12 weeks 18 weeks 24 weeks 32 weeks Optimal No.Diagnosis Therapy cycle evaluation evaluation evaluation evaluationevaluation efficacy 011 hepatocellular Second-line 15 SD reduced PR PRPR NA PR carcinoma therapy 014 hepatocellular Third-line 14 SD reducedSD reduced SD Performed, NA SD carcinoma therapy Not evaluated 019hepatocellular Second-line 11 SD SD reduced SD NA NA SD carcinomatherapy 021 hepatocellular First-line 9 SD increased SD Performed, NA NASD carcinoma therapy Not evaluated 027 hepatocellular Second-line 3 SDreduced Not performed NA NA NA SD carcinoma therapy 018 hepatocellularSecond-line 4 SD increased PD NA NA NA PD carcinoma therapy 016 gastriccancer Fouth-line 9 PR PD PD NA NA PR therapy 025 gastric cancerSecond-line 8 PR PR NA NA NA PR therapy 012 gastric cancer Multi-line 9SD reduced SD reduced SD increased NA NA SD therapy 013 gastric cancerThird-line 5 SD NA NA NA NA SD therapy 022 gastric cancer Second-line 10SD reduced SD SD NA NA SD therapy 024 gastric cancer Third-line 6 SDincreased PD NA NA NA SD therapy 026 gastric cancer Third-line 8 SDreduced SD NA NA NA SD therapy 028 gastric cancer Third-line 7 SDPerformed, NA NA NA SD therapy Not evaluated 015 gastric cancerSecond-line 5 9 weeks PD PD NA NA NA PD therapy 017 gastric cancerFifth-line 4 PD PD NA NA NA PD therapy 023 gastric cancer Second-line 5PD PD NA NA NA PD therapy 029 gastric cancer Multi-line 5 Performed, NANA NA NA To be therapy Not evaluated evaluated 030 gastric cancerThird-line 5 Performed, NA NA NA NA To be therapy Not evaluatedevaluated 031 gastric cancer Second-line 3 Not NA NA NA NA Not evaluatedtherapy Performed

TABLE 5 Efficacy evaluation of enrolled patients Screening Efficacyevaluation-Diameter(mm)/Baseline ratio (%) General No. Baseline 2 cycles4 cycles evaluation 01001 28   27/−3.6% 15.5/−44.6% PR 01002 20.6    1cycle, PD hydrothorax increased 01003 76.7 New onset of hydrothorax74.7/−2.6% SD 01005 122.9 liver metastases increased, PD enlarged 01006137   118/−13.9%  107/−22% SD 01007 134.2 117.7/−12.2%  107/−20.3 SD01008 58.3   52/−10.8% SD SD 01010 11    9/−20% SD 01011 36 SD SD 0101387 SD SD 01014 85.5 SD SD 01015 SD SD

We claim:
 1. A method for treating cancer in a subject in need thereof,the method comprising administering to the subject, an anti-PD-1antibody and apatinib or a pharmaceutically acceptable salt thereof,wherein the anti-PD-1 antibody comprises: an antibody light chainvariable region comprising LCDR1, LCDR2 and LCDR3 having the amino acidsequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively;and an antibody heavy chain variable region comprising HCDR1, HCDR2 andHCDR3 having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 andSEQ ID NO: 3, respectively, wherein the apatinib or the pharmaceuticallyacceptable salt thereof is administered orally at a dose from 125 mg to375 mg.
 2. The method of claim 1, wherein the pharmaceuticallyacceptable salt of apatinib is mesylate salt of apatinib orhydrochloride salt of apatinib.
 3. The method of claim 1, wherein theanti-PD-1 antibody is a humanized antibody.
 4. The method of claim 3,wherein the humanized antibody comprises an antibody light chainvariable region having the amino acid sequence of SEQ ID NO: 8 or a SEQID NO: 8 mutant having 1 to 10 amino acid substitutions in SEQ ID NO: 8,and an antibody heavy chain variable region having the amino acidsequence of SEQ ID NO: 7 or a SEQ ID NO: 7 mutant sequence having 1 to10 amino acid substitutions in SEQ ID NO:
 7. 5. The method of claim 1,wherein the antibody light chain variable region comprises the aminoacid sequence of SEQ ID NO: 8 and the antibody heavy chain variableregion comprises the amino acid sequence of SEQ ID NO:
 7. 6. The methodof claim 4, wherein the antibody light chain variable region comprises amutant sequence of the amino acid sequence of SEQ ID NO: 8 having theamino acid substitution A43S, and the antibody heavy chain variableregion comprises a mutant sequence of the amino acid sequence of SEQ IDNO: 7 having the amino acid substitution G44R.
 7. The method of claim 1,wherein the cancer is selected from the group consisting of breastcancer, lung cancer, liver cancer, gastric cancer, intestinal cancer,renal cancer, melanoma and non-small cell lung cancer.
 8. The method ofclaim 7, wherein the subject has failed at least one chemotherapy priorto the administration of the anti-PD-1 antibody and apatinib or thepharmaceutically acceptable salt thereof.
 9. The method of claim 1,wherein the anti-PD-1 antibody is administered at a dose of 2 mg/kg to 6mg/kg body weight of the subject or from 100 mg to 1000 mg peradministration.
 10. The method of claim 9, wherein the anti-PD-1antibody is administered at a dose from 200 mg to 600 mg peradministration.
 11. The method of claim 1, wherein the anti-PD-1antibody is administered at a dose of 3 mg/kg body weight of the subjector 200 mg once every 1 to 3 weeks.
 12. The method of claim 1, whereinthe anti-PD-1 antibody is administered at a dose of 3 mg/kg body weightof the subject or 200 mg once every two weeks.
 13. The method of claim1, wherein the apatinib or the pharmaceutically acceptable salt thereofis administered orally at a dose of 125 mg, 250 mg, or 375 mg.
 14. Themethod of claim 1, wherein the apatinib or the pharmaceuticallyacceptable salt thereof is administered orally at said dose of 125 mg to375 mg once daily.
 15. The method of claim 1, wherein the anti-PD-1antibody is administered at a dose of 3 mg/kg body weight of the subjector 200 mg.
 16. The method of claim 1, wherein the anti-PD-1 antibody isadministered at a dose of 3 mg/kg body weight of the subject or 200 mgonce every 1 to 3 weeks, and the apatinib or the pharmaceuticallyacceptable salt thereof is administered orally at said dose from 125 mgto 375 mg once daily.
 17. The method of claim 1, wherein the anti-PD-1antibody is administered at a dose of 3 mg/kg body weight of the subjector 200 mg once every two weeks, and the apatinib or the pharmaceuticallyacceptable salt thereof is administered orally at a dose of 250 mg or375 mg once daily.